Imidazole and imidazoyl containing compounds have properties that make them useful in pharmaceutical formulations; for example, imidazole is among the agents which exert a therapeutic effect in alleviating inflammation and tissue injury arising from physiological stress and mediated by prostaglandin end products.
Physiological stress results in an inflammatory response, accompanied by increased prostaglandin synthesis. Phagocytosis of inert particles, bacteria and viruses induces the release of inflammatory hormones and prostaglandins. The stress provoked by reduced oxygen tension, interrupted blood flow, or infections, is often accompanied by increased synthesis of prostaglandins, including the thromboxanes, from arachidonic acid. This synthesis, which occurs primarily in macrophages, begins with the conversion of free arachidonic acid to an alicyclic endoperoxide prostaglandin intermediate, PGH.sub.2, by means of a cyclo-oxygenase enzyme. PGH.sub.2 is the precursor for several end product prostanoids. Among these are prostacyclin, PGI.sub.2, whose formation is catalyzed by prostacyclin synthetase; the active prostaglandins PGF.sub.2a, PGE.sub.2, and PGD; and the thromboxane, TxA.sub.2, whose formation is catalyzed by thromboxane synthetase. Imidazole inhibits thromboxane synthetase and prevents the synthesis of TxA.sub.2 ; other agents, such as aspirin-like compounds, ibuprofen and indomethacin, inhibit cyclo-oxygenase, thus preventing the synthesis of all the prostanoids.
The prostaglandin end products have several various effects. They can act themselves to alter the pain threshold and, in a manner similar to mediators of the allergic response, to increase capillary permeability. They can also act on cells, primarily macrophages and platelets, to release substances that mediate an inflammatory response, which can involve infiltration of tissues with neutrophils, deposition of immune complexes, edema, and pain.
PGI.sub.2 and TxA.sub.2 are the most biologically active of the prostanoids, but exert opposite vasoactive and hemodynamic effects. For example, PGI.sub.2 is a potent vasodilator and inhibitor of platelet aggregation, whereas TxA.sub.2 is a vasoconstrictor and powerful inducer of platelet aggregation. Platelet aggregation results in an inflammatory response, thrombosis, and the unfavorable consequences. In the kidney, for example, thrombosis is followed by glomerulosclerosis, hypertension and cardiac hypertrophy. Imidazole, by selectively inhibiting thromboxane synthetase, shunts the conversion of PGH.sub.2 from TxA.sub.2 to PGI.sub.2, produces physiologically beneficial effects, and reduces tissue damage.
The effect is well demonstrated in the kidney, in which the tissue injury associated with inflammation consists of destruction of the glomerular capsule resulting from vasodilation, followed by deposition of fibrous material. Zipser, R. et al., Circulation Research 47(2): 231-237 (1980), found exaggerated prostaglandin and thromboxane production in the ex vivo perfused kidney, under conditions of renal vein constriction, which could be inhibited by preincubation with imidazole. Saito, H. et al., Nephron 36:38-45 (1984), studied the inhibition of inflammatory damage in the kidney by a specific thromboxane A2 synthetase inhibitor, 1-benzylimidazole (BIm). This agent delayed the progression of glomerulonephritis and in the tissues of treated animals platelet aggregation and fibrin deposits were reduced. Okegawa, T. et al. J. of Pharmacology and Experimental Therapeutics 225(1):213-218 (1983) noted that in endotoxic shock, toxin-stimulated macrophages infiltrate into tissues where they synthesize arachidonate metabolites which modulate an inflammatory response comprising vasodilation, edema and pain. A comparative study of the effectiveness of inflammation suppression by imidazole, indomethacin, and ibuprofen by Schirmer, W et al. Current Surgery March-April, 1987, pp. 102-105) in a model system of acute peritoneal sepsis indicated that imidazole, which inhibits only the formation of thromboxane A2, maintained its activity in preventing endotoxic shock over a longer period of time than the other two agents which act to inhibit the formation of all prostaglandins.
Histidine appears to have a protective effect on fatty acids, which are often present in the emulsifying agents of pharmaceutical preparations. Many medically useful agents are used parenterally in the form of emulsions, comprising a hydrophobic phase dispersed in an aqueous system. The emulsifying agent is commonly a phospholipid, which, because of its amphipathic nature, is able to form a stable association with both phases. Phospholipids are composed of a glycerol group to which fatty acids are attached by ester bonds. Emulsions may, in addition, comprise other types of lipids, some of which also contain fatty acids.
Parenteral emulsions present a particularly difficult buffering problem when they have an emulsifying agent such as phospholipids which comprises fatty acids. Phospholipids and other molecules comprising fatty acids may generate acidic substances after incorporation into emulsions, either while in storage or in use. The acidic substances may be free fatty acids and lysophosphatide species produced either from the hydrolysis of the ester bonds between the glycerol and fatty acid components of phospholipids, or similar free fatty acids resulting from the oxidation of unsaturated fatty acids at the site of their double bonds. Histidine, together with its structurally related amino acid, tryptophan, and sulfur-containing methionine, appears to be responsible for the ability of some proteins to quench singlet oxygen produced by photosynthetic processes and by this means to prevent their own oxidative decomposition. The rate of physical quenching is the same for both free amino acids and those incorporated into a protein structure. (Matheson, R. et al., Photochemistry and Photobiology, 21:165-171 (1975)). It is quite probable that histidine would be similarly effective in preventing the oxidative degradation of phospholipid fatty acids (or free fatty acids) which leads to acid generation and a decrease in pH in fluorocarbon emulsions.
With the exception of the stomach, the pH range in the body is maintained, through chemical exchanges occurring principally in the renal and respiratory systems, at about 6.4 to 7.5 for the tissues and between 7.35 and 7.45 for the extracellular fluids. Most physiological processes and biochemical reactions occur then within relatively narrow pH limits. Little is known about the rates of enzyme reactions at physiological pH. The physiological pH restriction may provide an optimum pH range for the rates of certain enzyme reactions, and may act as a control mechanism for those for which that pH is not optimum.
It is clear, however, that processes in some tissues and organs are sensitive to small changes in pH. The effect of an acid environment on the heart is an example. As the pH of arterial blood falls, the coronary vessels dilate to increase blood flow and a supply of oxygen to the cardiac muscle; at a critically low pH, there is a sudden, sharp decrease in myocardial contractility. In dogs, the lethal pH is 6.0, at which point there is cardiac arrest in extreme diastole. The effect is independent of the nature of the anion species of the acid. Another example is an effect such as occurs in metabolic alkalosis, wherein an increase in the pH of the blood moves extracellular fluid into the cells, resulting in tissue edema.
It is important then that substances introduced parenterally into the body, and particularly those placed in the circulation, most critically the cardiac circulation, are buffered to a physiological pH, so as not to disturb the biochemical balance. It is further important that the agents used to buffer these substances are physiologically innocuous.
The natural physiological buffering agents are principally carbonate and phosphate ions and the amino acids. In the blood the buffers NaHCO.sub.3 /H.sub.2 CO.sub.3 along with constituent blood proteins hemoglobin, oxyhemoglobin, albumin and globulin and Na.sub.2 HPO.sub.4 /NaH.sub.2 PO.sub.4 resist changes in pH. Proteins may contain groups which act as buffers, depending on the pH; these are commonly the carboxyl groups of glutamic and aspartic acids and the amino groups of lysine, the guanido group of arginine and the imidazolyl group of histidine.
Because of their natural buffering capacity, carbonates and phosphate salts are commonly used as buffers in intravenous solutions. A difficulty with these buffers that becomes more significant in highly buffered solutions, is that both carbonate and phosphate ions form insoluble precipitates with divalent cations such as magnesium and calcium. This has two undesirable effects. First, it reduces cation concentrations and inhibits critical metabolic processes, such as Mg.sup.++ dependent enzyme reactions, and Ca.sup.++ dependent muscular contractions. Secondly, the insoluble precipitates formed can block the blood flow through small vessels, and those formed when solutions are introduced into cavities such as the brain ventricles can deposit a film on internal membrane surfaces which will then interfere with membrane transport.
Amino acids may be advantageously used as buffers in pharmaceutical applications because they have neither of these undesirable actions, and they are, in addition, metabolizable, nutritive substances.
Histidine appears to act as a natural buffer in tissue such as muscle where high metabolic activity occurs. The process of anaerobic glycolysis necessary for rapid ATP production in exerted muscle is associated with elevated lactate, proton accumulation and a fall in pH. Abe, H. et al., Am. J. Physiol. 249:R449-R454 (1985), found that histidine and histidine-containing dipeptides are the principal buffers in the skeletal muscle of fish such as marlin that has a high activity pattern, and that the histidine buffered tissue had a higher buffering capacity than corresponding tissue from less active fish. In a parallel study on humans, Parkhouse, W. S. et al., J. Appl. Physiol. 58(1):14-17 (1985). found higher buffering capacity and higher levels of histidine related peptides in needle biopsies of muscle tissue from athletes capable of superior high-intensity running performance.
Histidine has been used as a buffer for cardioplegic solutions which are used to preserve organs such as hearts and kidneys during surgical procedures in which ischemic intervals occur and during transplant procedures. Bretschneider U.S. Pat. No. 4,415,556. The composition of these solutions has been determined empirically to comprise substances normally found in blood and electrolytes favorable to preserving the contractility of cardiac muscle and preventing edema. Histidine content is adjusted so that the solutions provide the pH and pH buffering capacity of cardiac tissue as determined by comparative manual titrations. (Kresh, J. Y. et al., J. of Thoracic and Cardiovascular Surgery, 93(2):309-311 (1987)).
One problem in buffering lipid containing emulsions is that acid generating oxidative processes, which occur to an unpredictable extent, may exceed the buffering capacity of the solution, and bring the pH down to unacceptably low levels.
One approach to this problem has been to prepare an emulsion and add sufficient sodium hydroxide or other inorganic base to the preparation to achieve an initial pH high enough to anticipate a degree of acid generation. However, this method of protection, high formulation pH, can actually accelerate the degradation of the lipids and aggravate the problem it is intended to correct.
Presuming one could predict the amount of expected proton generation in a lipid-containing emulsion, it would still not be possible to provide the necessary buffering capacity to the emulsion accurately enough so as to assure its pH stability within an acceptable range using present methods.
The major difficulty is that there is no theoretical mathematical expression for the titration curve of a polyprotic acid or base, and hence no way to calculate the buffer capacity of these substances exactly. The present method of calculating buffer capacity depends on the use of the Henderson-Hasselbach equation. This expression, ##EQU1## relates the pH to the ionization constant for a single ionizable group of a weak acid or base. Buffer capacity can be determined for that ionizable group by calculating the change in the log Ac.sup.- /HAc term corresponding to a change in the pH. One of the shortcomings of this method is that it can only provide an approximation of the buffering capacity of a polyprotic buffer or a mixture of buffers having different pK.sub.a 's, because the buffering capacity of each ionizable group must be calculated independently, and the interaction between these groups ignored. When the pK.sub.a 's are quite different, the error is negligible (but not zero); but when they are close in value, the mis-estimation is serious.
The pK is that pH at which a weak acid or base is 50% converted to its salt, and it is at this point that its buffering capacity, the amount of H.sup.+ or OH.sup.- ions it can neutralize per unit change in pH, is highest. Histidine is one of the most effective amino acid buffers for parenteral use because it contains an imidazole ring having a nitrogen site with a pK of about 6.83, near the center of the physiological pH range. Imidazole itself, in which the pK at the nitrogen site is about 6.99, also acts as a buffer.
A determination of the optimal agent for providing the required buffer capacity by the trial and error process of manual titration would be tedious and expensive and particularly difficult if a combination of amino acids were used.
Further, an exact determination of the necessary buffering capacity in the emulsion must take into consideration the influence of the ionizable groups of the phospholipid species present. Although the effect of these groups is negligible at neutrality, having pK.sub.a 's of 1-2 and 10-13 for most species, the effect is also not zero. These effects cannot be determined manually, because the titration of the buffer with acid or base would hydrolyze the lipid components, generating interfering acid or base.
The emulsions cannot be adequately buffered simply by adding an excess of an amino acid buffer, because the constraint that all administered parenteral agents must be isotonic limits the amount of amino acid that can be added to the emulsion. For that reason it is important to determine precisely the effective amount of these agents in the emulsions.
Amino acids such as histidine and other structures containing an imidazolyl group appear to be particularly appropriate buffering agents for fluorocarbon emulsions. Moreover, the anti-inflammatory properties of imidazole and the anti-oxidative properties of histidine can provide further advantages when incorporated in fluorocarbon formulations.
Fluorocarbon emulsions can be widely used intravascularly and extravascularly as contrast agents. Further, fluorocarbons have the capacity to dissolve high concentrations of oxygen, making them suitable as artificial blood and as oxygen transport agents in the treatment of local ischemias. However, the intravascular administration of fluorocarbon emulsions is frequently accompanied by a transient fever and inflammatory response. I have found that agents such as imidazole, ibuprofen and indomethacin have an anti-pyretic effect which mitigates the inflammatory response when they are present in these emulsions.
For all the foregoing reasons, amino acids such as histidine and its constituent imidazolyl group are believed to be particularly effective pharmacological components as well as appropriate buffering agents for fluorocarbon emulsions.
It is therefore an object of the invention to provide formulations for fluorocarbon emulsions comprising an effective therapeutic concentration of imidazole or related pharmacologically active agents.
It is desirable to provide formulations for fluorocarbon emulsions which are protected against decomposition by the use of effective buffers and antioxidants. It is therefore further an object of the invention to provide formulations for fluorocarbon emulsions comprising effective buffering concentration of amino acids, and particularly histidine and related compounds comprising an imidazolyl group.
It is further an object of the invention to provide a method for selecting buffers for formulations of fluorocarbon emulsions, comprising amino acids which provide a known buffering capacity for those emulsions within a given pH range.
It is further an object of the invention to provide formulations of amino acid buffered fluorocarbon emulsions which are stable within a physiological pH range during a sterilization process and for an estimated period of storage.